Normal genes (DNA) encode proteins necessary for the growth, differentiation and survival of cells. Overexpression, mutation or expression of normal proteins at an inappropriate time in the cell cycle can transform normal cells to cancer cells. When normal genes act in this manner they are referred to as oncogenes.
Ras genes are found in a wide variety of nucleated mammalian cells and participate in normal cellular functions. The family of ras genes encode a series of immunologically related proteins with a molecular weight of 21,000 and are referred to as p21s. Ras genes present in mammalian cells have been demonstrated to be homologous to murine sarcoma viral oncogenes. (Weinberg, et al., U.S. Pat. No. 4,535,058: Harvey (1964), Nature. 104:1104: Kirsten et al. (1967). J.N.C.I., 38:311). Viral and cellular ras genes encode membrane bound proteins (Willingham, et al. (1980), Cell. 19:1005) which bind quanine nucleotides (Scolnick, et al. (1979) PNAS (USA), 76:5355: Pageorge, et al. (1982), J. Virol., 44:509: and Finek, et al. (1984), Cell. 37:151) and possess intrinsic GTPase activity (McGrath et al. (1984). Nature, 310:644: Sweet et al. (1984). Nature, 311:273; Gibbs et al. (1984) PNAS (USA) 81:5704; and Manne et al. (1985) PNAS 82:376).
DNA mediated transfection experiments using NIH3T3 cells as recipients have led to the identification of a family of activated transforming genes homologous to the ras genes of the Harvey (ras-H) and Kirsten (ras-K) sarcoma viruses. A third member of the ras family designated ras-N has been identified but has not been found to have a retroviral counterpart. Activated ras genes are structurally distinct from their normal homologs, having amino acid substitutions in the protein at positions 12,13 or 61. (Tabin, et al. (1982), Nature, 300:143: Reddy et al. (1982) Nature, 300:149; Bos et al. (1985) Nature, 315:716; and Yuasa et al. (1983) Nature, 303:775-779. Taparowsky et al., Banbury Report, 14:123-133 (1983) cited in Chem. Abstracts, CA 100(1):1425n, teaches that the change at residue 12 from N-terminus of the H ras p21 from glycine to valine is sufficient to convert the normal gene to a transforming gene. Shimizu et al., Nature, 304 (5926), 497-500 (1983) cited in Chem. Abstracts, 99(19):1530936, teaches the presence of a cysteine residue at amino acid 12 in the human lung cancer cell line calu-1 homolog of the v-ki-ras gene. Fasano et al., J. Mol. Appl. Genet., 2(2):173-180 (1983) cited in Chem. Abstracts CA, 99(19):153080v, teaches that the T24H-ras-1 gene product is nearly identical to the v-H-ras p21 transforming protein encoded by Harvey sarcoma virus. Activated ras transforming genes have been found in 10-20% of neoplasms including sarcomas, neuroblastomas, melanomas and carcinomas. In certain forms of leukemia activated ras genes and the corresponding proteins have been found in over 50% of the tumors studied. These activated ras gene and mutated proteins have also been found in established cell lines as well as primary and metastatic tumors. Gambke et al. (Nature 307:476, 1984) demonstrated a transforming N-ras gene in bone marrow cells from a patient with acute myeloblastic leukemia (AML). In contrast DNA from fibroblast cells from the same patient was not transforming.
The p21 ras protein in its normal nonactivated form contains the glycine amino acid at positions 12 and 13 and the glutamine amino acid at position 61. The p21 protein found in normal cells has the following primary amino acid structure for amino acid residues 5 through 19: .sup.5 Lysine-Leucine-Valine-Valine-Valine-Glycine-Alanine-Glycine-Glycine-Valine -Glycine-Lysine-Serine-Alanine-Leucine.sup.19.
Previous reports (Furth et al. (1982), J. Virol., 43:294) have described several rat monoclonal antibodies reactive with normal and activated or oncogenic (mutated) ras p21 proteins in yeast and mammalian cells. Carney et al., Proc. Nat. Acad. Sci., USA, Vol. 83, pp. 7485-7489 (1986) and EPO Publication No. 019003 published on Aug. 6, 1986 disclose a monoclonal antibody specific for an activated ras protein. This monoclonal antibody was raised against a synthetic peptide corresponding to amino acids of a mutated ras gene encoding valine instead of glycine at position 12. Carney et al., UCLA Symp. Mol. Cell. Biol., New Ser. 1985 cited in Chem. Abstracts 104:1665706, disclose a monoclonal antibody raised against a ras related synthetic peptide showing immunoreactivity with human carcinomas. Carney et al. reported a series of monoclonal antibodies raised against synthetic peptides containing amino acid substitutions of glutamic acid, arginine and valine at position 12 (A Book of Abstracts from the 3d Annual Meeting on Oncogenes held at Hood College, Frederick, Md., Jul. 7-11, 1987). Other monoclonal antibodies generated by various methods have also been reported to react with the various forms of the ras p21 protein. Hand et al. Proc. Nat. Acad. Sci. USA, Vol. 81, pp. 5227-5231 (1984); Thor et al., Nature, Vol. 311, pp. 562-565 (1984); Wong et al., Cancer Research, Vol. 46, pp. 6029-6033 (1986), and Tanaka, Proc. Natl. Acad. Sci., USA, Vol. 82, pp 3400-3404 (1985).
Several scientific reports have shown that normal cells contain ras proteins with glycine at position 13.
In 1985 Bos et al. (Nature 315:726 1985) demonstrated that DNA isolated from cells of AML patients were able to transform NIH3T3 cells. This result is indicative and highly suggestive for the presence of an oncogene. These transforming genes were shown to be activated ras genes. In contrast DNA from normal tissues were non-transforming and therefore did not contain activated N ras. These investigators analyzed the activated N ras genes for the presence of mutations using oligonucleotide probe and found that the activated N ras genes contain mutations that result in amino acid substitutions at position 13 of protein. These mutations at position 13 were shown to be either aspartic acid or valine instead of the normal amino acid glycine.
Two reports in 1987 described ras mutations with arginine at position 13. Nitta et al. have shown (Jpn J Cancer Res. (Gann), 78,21-26 1987) an amino acid substitution of arginine for glycine at position 13 of an activated N ras p21 isolated from a human rectal carcinoma. A report by Hirai et al. (Nature 327:430 1987) has shown activated N ras genes in bone marrow cells from patients with myelodysplastic syndrome. The observations made by Hirai et al. suggests that the presence of activated N ras genes with position 13 mutations may be important in early stages of leukemia.
A report by E. Liu et al. (Nature 330:186, 1987) demonstrated the presence of the aspartic acid mutation at position 13 of the ras p21 in a patient with myelodysplastic disease 1.5 years before the patient progressed to acute leukemia. Thus screening patients with myeloplastic syndrome for the presence of activated ras proteins with position 13 mutations with monoclonal antibodies that are the subject of this invention may be a valuable test to predict which patients with myeloplastic syndrome have an increased risk of developing acute leukemia.
Most recently Wodnar-Filipowica et al. reported (Oncogene, pp. 457-461, Vol. 1., No. 4 (1987) the presence of activated N ras genes in a human T cell non-Hodgkin's lymphoma. These studies demonstrated the substitution of cysteine for glycine at position 13.